Functional Tumor Targeting Nano‐Systems for Reprogramming Circulating Tumor Cells with In Situ Evaluation on Therapeutic Efficiency at the Single‐Cell Level

Abstract Tumor heterogeneity is primarily responsible for treatment resistance and cancer relapses. Being critically important to address this issue, the timely evaluation of the appropriateness of therapeutic actions at the single‐cell level is still facing challenges. By using multi‐functionalized nano‐systems with the delivery vector composed of histone for plasmids loading, hyaluronic acid for tumor targeting, and a fusion peptide for C‐X‐C motif chemokine receptor 4 (CXCR4） targeting as well as nuclear localization, the reprogramming of circulating tumor cells (CTCs) with in situ detection on biomarkers at the single‐cell level is realized. By efficient co‐delivery of the genome editing plasmid for CXCR4 knockout and molecular beacons for detection of upregulated mRNA biomarkers into CTCs in unprocessed whole blood, the therapeutic outcomes of genome editing at the single‐cell level can be in situ evaluated. The single‐cell analysis shows that CXCR4 in CTCs of cancer patients is efficiently downregulated, resulting in upregulated anticancer biomarkers such as p53 and p21. The study provides a facile strategy for in‐depth profiling of cancer cell responses to therapeutic actions at single‐cell resolution to evaluate the outcomes of treatments timely and conveniently.


Characterizations of plasmid and/or molecular beacon delivery systems
The size and ζ potential of nanoparticles were measured by a Zetasizer (Nano ZS, Malvern Instruments). Before the measurement, 900 µl of ultrapure water was added in the ultrapure water containing nanoparticles (100 µl) for dilution.
To determine the encapsulation efficiency of plasmids and/or molecular beacons, 900 µl of ultrapure water was added in the ultrapure water containing nanoparticles (100 µl) for dilution, and then the sample was centrifuged at 10000 rpm for 1 h. The unencapsulated free plasmids and/or molecular beacons in the supernatants were measured using a Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) according to the manufacturer's protocol.
Fluorescence spectroscopy (RF-5301 PC, Shimadzu) was used to determine the fluorescence intensity. The encapsulation efficiency of plasmids and/or molecular beacons was calculated as encapsulation efficiency = (MT -MF) / WT × 100% where MT was the total mass of plasmid and/or molecular beacon and MF is the mass of unencapsulated free plasmid and/or molecular beacon.
To evaluate the assembly efficiency of T22-NLS peptide, FITC labeled T22-NLS was used to prepared the plasmid loaded nanoparticles. 900 µl of ultrapure water was added in the S5 ultrapure water containing nanoparticles (100 µl) for dilution, and then the sample was centrifuged at 10000 rpm for 1 h. The free FITC labeled peptide in the supernatant was measured with fluorescence spectroscopy Shimadzu). The results showed about 70% of FITC labeled T22-NLS was self-assembled onto CRISPR-Cas9 plasmid@histone/FITC labeled peptide/hyaluronic acid nanoparticles.
The nanoparticles supported on a 200-mesh copper grid and stained with phosphotungstic acid were observed by transmission electron microscopy (JEM-2100).

Cellular internalization study
CRISPR-Cas9 plasmid was labeled by YOYO-1, and the YOYO-1 labeled plasmid was used to prepared plasmid loaded nanoparticles. For flow cytometry analysis, cells were seeded in a 6-well plate (2×10 5 cells in 2 ml of DMEM per well). After incubation for 24 h, the culture medium was removed, and the fresh medium containing nanoparticles loaded with YOYO-1 labeled plasmid was added to the cells. After co-incubation with the plasmid loaded nanoparticles at a plasmid concentration of 2 μg ml -1 for 4 h, the cells were washed thrice by PBS, digested with trypsin, collected by centrifugation, resuspended in 200 μl of PBS, and analyzed by flow cytometry (Dakewe EXFLOW-206).
For confocal laser scanning microscopy (CLSM), cells were seeded in a glass-bottomed culture dish (35 mm) (1×10 5 cells in 1 ml of DMEM). After incubation at 37 °C for 24 h, the culture medium was removed, and the fresh medium containing nanoparticles loaded with YOYO-1 labeled plasmid was added to the cells. After co-incubation with the plasmid loaded nanoparticles at a plasmid concentration of 2 μg ml -1 for 4 h, the cells were washed thrice by PBS. The cell nuclei were stained by DAPI for 5 min, and then the cells were observed by CLSM at 400× magnification (PerkinElmer UltraVIEW VoX). To observe the endosomal escape ability, the cells were co-incubated with the nanoparticles loaded with YOYO-1 labeled plasmid for 2, 4, and 6 h, respectively, and then the cells were washed thrice by PBS, stained by LysoTracker Red (Invitrogen) for 45 min, and then stained by Hoechst 33342 for 15 min at 37 °C. After that, the cells were washed by PBS, and observed by CLSM at 600× magnification.

MTT assay
To evaluate cell viability, cells were seeded in a 96-well plate (5×10 3 cells in 100 μl DMEM per well). After incubation for 24 h, the medium was replaced by fresh medium containing plasmid loaded nanoparticles. After the cells were treated with plasmid loaded nanoparticles S6 at a plasmid concentration of 2 μg ml -1 for 48 h, MTT (5 μg μl -1 , 10 μl) was added in every well. After 4 h, the supernatant was removed, and 200 μl of DMSO was added in each well to dissolve the formazan crystals. The absorbance of the solution was measured at 570 nm by a microplate reader (Bio-Rad 550) to determine the OD value. The cell viability was calculated as cell viability=ODtreated /ODcontrol ×100% where ODtreated was obtained from the cells treated by a particular agent and ODcontrol was obtained from the untreated cells.

Western blot assay
The proteins in the unedited cells and genome edited cells were studied by Western blotting as detailed below. Cells were seeded in a 6-well plate (2×10 5 cells in 2 ml DMEM per well).
After incubation for 24 h, the medium was replaced by fresh medium containing plasmid loaded nanoparticles, and the cells were treated with plasmid (CRISPR-Cas9 plasmid for genome editing, or control plasmid for comparison) loaded nanoparticles at a plasmid concentration of 2 μg ml -1 for 48 h. After that, the cells were washed with PBS triple times, lysed and suspended in sodium dodecyl sulfate (SDS) sample buffer containing 1% βmercaptoethanol. Total protein extracts were subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to poly(vinylidene fluoride) membranes (Millipore). To block non-specific binding sites, the membranes were treated with TBST (Tris-buffered saline with Tween-20) containing 5% milk for 1 h. Then the membranes were incubated with the primary antibody overnight at 4 °C. After washing, the membranes were incubated with the secondary antibody for 1 h. Then an enhanced chemiluminescence system (Aspen) was used to detect the signals.
CXCR4 and CD44 in native HeLa and 293 cells were analyzed by Western blotting by using the cells without any treatment.

Quantitative polymerase chain reaction (qPCR) assay
The levels of CXCR4 mRNA, p53 mRNA, p21 mRNA, microRNA-21 and microRNA-221 in genome edited cells and unedited cells were analyzed by qPCR. Cells were seeded in a 6-well plate (2×10 5 cells in 2 ml DMEM per well). After incubation for 24 h, the medium was replaced by fresh medium containing plasmid (CRISPR-Cas9 plasmid for genome editing, or control plasmid for comparison) loaded nanoparticles, and the cells were treated with plasmid loaded nanoparticles at a plasmid concentration of 2 μg ml -1 for 48 h. After that, the cells were S7 collected and the total RNA was extracted with a High Pure RNA Isolation Kit (Invitrogen).
An EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotech) was used for the first cDNA strand synthesized, and then qPCR was performed on a StepOne Real-Time PCR system (Life Technologies) with EnTurbo™ SYBR Green PCR SuperMix (ELK Biotech). The relative RNA levels were measured by the 2 -ΔΔCt method.

T7 endonuclease I (T7E1) assay
Cells were seeded in a 6-well plate (2×10 5 cells in 2 ml DMEM per well). After incubation for 24 h, the medium was replaced by fresh medium containing plasmid loaded nanoparticles, and the cells were treated with CRISPR-Cas9 plasmid loaded nanoparticles at a plasmid concentration of 2 μg ml -1 for 48 h. After that, the genomic DNA was extracted from the cells using QuickExtract DNA extraction solution (Epicentre). Genomic regions of CXCR4 gene were amplified by PCR and then the homoduplex products of PCR were denatured, rehybridized under stepdown annealing conditions to generate homo-and heteroduplexes. The mixture of duplexes was digested with T7E1 (NEB) that can cleave heteroduplex DNA mismatched. The products were electrophoresed on a 2% agarose gel. For comparison, the extracted genomic DNA without denaturation and rehybridization was also treated by T7E1 to exclude false positive results.

DNA sequencing assay
Cells were seeded in a 6-well plate (2×10 5 cells in 2 ml DMEM per well). After incubation for 24 h, the medium was replaced by fresh medium containing P@HPH, and the cells were treated with P@HPH at a plasmid concentration of 2 μg ml -1 for 48 h. After that, the genomic DNA was extracted from the cells and amplified by PCR. The product of PCR was subjected to TA cloning. The colonies were sequenced using an 3730XL DNA analyzer (Applied Biosystems).

Analysis on CXCR4 labeled by antibody in cancer cells
Cells were seeded in a 6-well plate (2×10 5 cells in 2 ml of DMEM per well). After incubation for 24 h, the medium was replaced by fresh medium containing plasmid loaded nanoparticles.
After the cells were treated with plasmid loaded nanoparticles at a plasmid concentration of 2 μg ml -1 for 48 h, the medium was removed. The cells were labeled by the anti-human PE conjugated CXCR4 antibody (200 μg ml -1 , 5 μl) (Biolegend) and then incubated on ice in the dark for 20 min, harvested and analyzed by flow cytometry. For comparison, untreated cells S8 were used as a control, and mouse IgG2a kappa isotype control (Biolegend) was also analyzed to exclude false positive cells.

Cell apoptosis assay
Cells were seeded in a 6-well plate (2×10 5 cells in 2 ml DMEM per well). After incubation for 24 h, the medium was replaced by fresh medium containing plasmid (CRISPR-Cas9 plasmid for genome editing, or control plasmid for comparison) loaded nanoparticles, and the cells were treated with plasmid loaded nanoparticles at a plasmid concentration of 2 μg ml -1 for 48 h. After that, the cells were collected and stained by an Annexin V-FITC/PI Staining Assay Kit (4A Biotech Co. Ltd., China) based on the manufacturer's protocol. The stained cells were analyzed by flow cytometry (Dakewe EXFLOW-206).

Wound healing assay
Cells were seeded in a 6-well plate (2×10 5 cells in 2 ml DMEM per well). After incubation for 24 h, the medium was replaced by fresh medium containing plasmid (CRISPR-Cas9 plasmid for genome editing, or control plasmid for comparison) loaded nanoparticles, and the cells were treated with plasmid loaded nanoparticles at a plasmid concentration of 2 μg ml -1 for 48 h. After that, the cells were collected and seeded in a new 6-well plate (1×10 5 cells in 2 ml DMEM per well). When the cells were grown to 90% confluence, a 200 µl micropipette tip sterile was used to scratch a straight line. Then the debris was removed by PBS washing, and fresh DMEM (2% FBS) was added in the plate. After incubation for 24 h, the cells were washed twice by PBS, and observed by an inverted microscope (Olympus IX73) at 200× magnification.

Transwell invasion assay
Cells were seeded in a 6-well plate (2×10 5 cells in 2 ml DMEM per well). After incubation for 24 h, the medium was replaced by fresh medium containing plasmid (CRISPR-Cas9 plasmid for genome editing, or control plasmid for comparison) loaded nanoparticles, and the cells were treated with plasmid loaded nanoparticles at a plasmid concentration of 2 μg ml -1 for 48 h. After that, the cells were suspended in 200 μl of serum-free DMEM and seeded into the Matrigel pro-coated upper chamber (1×10 4 cells per well in a 24-well chamber). 600 µl of DMEM (10% FBS) was added to the lower chamber to form a chemoattractant environment.
After incubation for 24 h, the noninvasive cells remained on the upper chamber were removed by cotton swabs. The cells that invaded to the lower chamber were rinsed with PBS twice, S9 fixed with 4% paraformaldehyde for 20 min, and stained with 0.1% crystal violet for 10 min.
Subsequently, the invading cells were visualized by an inverted microscope (Olympus IX73) at 200× magnification.

Detection on CXCR4 mRNA in edited and unedited cancer cells by molecular beacon delivery systems
Cells were seeded in a glass-bottomed culture dish (35 mm) (1×10 5 cells in 1 ml DMEM).
After incubation for 24 h, the medium was replaced by fresh medium containing plasmid

Detection on p53 mRNA and p21 mRNA in edited and unedited cancer cells by plasmid/molecular beacon co-delivery systems
Cells were seeded in a glass-bottomed culture dish (35 mm) (1×10 5 cells in 1 ml DMEM).
After incubation for 24 h, the medium was replaced by fresh medium containing plasmid and molecular beacon co-loaded nanoparticles (P/MB2@HPH, CP/MB2@HPH, P/MB3@HPH, and CP/MB3@HPH, respectively), and the cells were treated with plasmid and molecular beacon co-loaded nanoparticles at a plasmid concentration of 2 μg ml -1 and a MB concentration of 0.1 nmol ml -1 for 48 h. After that, the cells were washed thrice by PBS, stained by DAPI for 5 min and observed by CLSM (PerkinElmer UltraVIEW VoX) at 400× magnification. S10 Cells were seeded in a 6-well plate (2×10 5 cells in 2 ml DMEM per well). After incubation for 24 h, the medium was replaced by fresh medium containing plasmid and molecular beacon co-loaded nanoparticles (CP/MB2@HPH, P/MB2@HPH, CP/MB3@HPH, P/MB3@HPH, respectively), and the cells were treated with plasmid and molecular beacon co-loaded nanoparticles at a plasmid concentration of 2 μg ml -1 and a MB concentration of 0.1 nmol ml -1 for 48 h. After that, the cells were washed with PBS thrice, digested with trypsin, collected by centrifugation, suspended in 200 μl of PBS, and subsequently analyzed by flow cytometry (Dakewe EXFLOW-206).

Blood sample collection
Peripheral blood samples were obtained from cancer patients in Affiliated Hospital of Anhui Medical University. The study was approved by the Ethics Committee of Anhui Medical University (Approval number: 2021H001). Informed written consent of all participants was obtained, and all the experiments were conducted following pertinent guidelines.

Evaluation of the stability of the delivery system in whole blood
CRISPR-Cas9 plasmid was labeled by TOTO-3. T22-NLS peptide was labeled by FITC. The TOTO-3 labeled plasmid and FITC labeled T22-NLS were used to prepared plasmid loaded nanoparticles (P@HPH).
The EDTA-anticoagulated whole blood (4 ml) from the patient was placed in a 6-well plate (2 ml of whole blood per well). Then P@HPH loaded with 4 μg of CRISPR-Cas9 plasmid in 200 μl of ultrapure water were added to 2 ml of whole blood. After 4 h, the blood in each well was mixed with 50 ml of PBS for dilution, and then filtered with a 7 µm pore sized membrane filter to remove blood cells. CTCs on the filter membrane were fixed with

CXCR4 in genome edited CTCs as compared with unedited CTCs
The EDTA-anticoagulated whole blood (4 ml from each patient) was placed in a 6-well plate (2 ml of whole blood per well). Then P@HPH loaded with 4 μg of CRISPR-Cas9 plasmid in S11 200 μl of ultrapure water was added to 2 ml of whole blood in one well, and CP@HPH loaded with 4 μg of control plasmid in 200 μl of ultrapure water was added to 2 ml of whole blood in the other well. After 12 h, the blood in each well was mixed with 50 ml of PBS for dilution, and then filtered with a 7 µm pore sized membrane filter to remove blood cells. Subsequently, CTCs on the filter membrane were placed in a 6-well plate and incubated in DMEM (2 ml per well) for 36 h. Then the culture medium was removed and then the fresh DMEM containing MB1@HPH was added and CTCs was co-incubated with MB1@HPH at a MB concentration of 0.1 nmol ml -1 for 4 h. After that, the culture medium was removed. CTCs on the filter membrane were fixed with 4% paraformaldehyde for 15 min, co-incubated with anti-

CTCs as compared with unedited CTCs by cancer-targeting plasmid/molecular beacon co-delivery systems
The EDTA-anticoagulated whole blood (4 ml from each patient) was placed in a 6-well plate (2 ml of whole blood per well). Then P/MB2@HPH or P/MB3@HPH nanoparticles loaded with 4 μg of CRISPR-Cas9 plasmid and 0.2 nmol of MB in 200 μl of ultrapure water were added to 2 ml of whole blood in one well, and CP/MB2@HPH or CP/MB3@HPH nanoparticles loaded with 4 μg of control plasmid and 0.2 nmol of MB in 200 μl of ultrapure water were added to 2 ml of whole blood in the other well. After 12 h, the blood in each well was mixed with 50 ml of PBS for dilution, and then filtered with a 7 µm pore sized membrane filter to remove blood cells. Subsequently, CTCs on the filter membrane were placed in a 6-S12 well plate and incubated in DMEM (2 ml per well) for 36 h. After that, the culture medium was removed. CTCs on the filter membrane were fixed with 4% paraformaldehyde for 15 min, co-incubated with anti-CK8/18/19 antibody (1:75 dilution) (Abcam) and the anti-

Evaluation on viability of CTCs after genome editing
CTCs were treated with P@HPH (in whole blood for 12 h and then in DMEM for 36 h). After that, the culture medium was removed, and CTCs were stained with Calcein AM (Yeasen, China) with a concentration of 151 μg ml -1 , and observed with an inverted microscope (Olympus IX73) at 600× magnification.

Statistical analysis
The measurements were performed in triplicate, and data are given as mean ± standard deviation (s.d.). Statistical analysis was performed with GraphPad Prism 8 software using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test or a Student's t test. P < 0.05 was considered statistically significant. S13    Unedited cells treated by the control plasmid delivery system (CP@HPH) were studied for comparison. Data are mean ± s.d, n=3. Statistical analysis was performed by using one-way ANOVA with Tukey's multiple comparison test. **P< 0.01, ***P< 0.001, ****P< 0.0001.
S22 Figure S11. Study on the stability of plasmid delivery system in whole blood containing CTCs. P@HPH was added in the whole blood from the patient BC0 followed by coincubation for 4 h. T22-NLS was labeled by FITC. CRISPR/Cas9 plasmid was labeled by TOTO-3. Cell nuclei were stained by DAPI. Scale bar: 15 µm. CTCs from the patient BC3. CTCs were treated by CP/MB3@HPH (unedited) or P/MB3@HPH (edited) for 48 h before antibody labeling. Fluorescence intensity of each CTC was analyzed by Volocity Demo 6.1.1 software. Statistical analysis was performed by using Student's t-test. **P<0.01, ****P<0.0001.